Extreme morphological and ecological homoplasy in tropical

Gabriela Parra-Olea* and David B. Wake†

Museum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720-3160

Contributed by David B. Wake, April 25, 2001 Fossorial salamanders typically have elongate and attenuated We analyzed sequences of mtDNA of many tropical bolito- heads and bodies, diminutive limbs, hands and feet, and extremely glossines, including all recognized genera, and determined that elongate tails. Batrachoseps from California, Lineatriton from east- Lineatriton and Oedipina are much more closely related to other ern Me´xico, and Oedipina from southern Me´xico to Ecuador, all taxa than to each other (3, 4). Not only was Lineatriton deeply members of the family , tribe Bolitoglossini, resem- nested within the large, mainly Mexican genus , ble one another in external morphology, which has evolved inde- but populations of Lineatriton from different parts of its geo- pendently. Whereas Oedipina and Batrachoseps are elongate be- graphic range were more closely related to different species of cause there are more trunk vertebrae, a widespread homoplasy Pseudoeurycea than to each other. Here we analyze molecular (parallelism) in salamanders, the genus Lineatriton is unique in data for 1,816 bp of mtDNA derived from three genes, reject the having evolved convergently by an alternate ‘‘giraffe-neck’’ de- monophyly of Lineatriton, and support an extraordinary case of velopmental program. Lineatriton has the same number of trunk homoplasy in a putative species that previously has been con- vertebrae as related, nonelongated taxa, but individual trunk sidered to be extremely specialized, and unique, in both mor- vertebrae are elongated. A robust phylogenetic hypothesis, based phology and ecology. on sequences of three mtDNA genes, finds Lineatriton to be deeply nested within a clade characterized by generalized ecology and Materials and Methods morphology. Lineatriton lineolus, the only currently recognized We collected specimens of all populations reported herein and taxon in the genus, shows unanticipated genetic diversity. Sur- preserved either frozen tissues or placed small pieces of tail prisingly, geographically separated populations of L. lineolus are tissue in 95% ethanol. The in-group includes eight samples of L. not monophyletic, but are sister taxa of different species of the lineolus from central western Veracruz, Me´xico, and four sam- morphologically generalized genus Pseudoeurycea. Lineatriton, ples from the Los Tuxtlas area of coastal southeastern Veracruz. long thought to be a unique monospecific lineage, is polyphyletic. The first of sequential out-groups includes samples of 15 species Accordingly, the specialized morphology of Lineatriton displays of Pseudoeurycea from Me´xico. More distant out-groups were homoplasy at two hierarchical levels: (i) with respect to other selected from other members (Batrachoseps and )ofthe elongate lineages in the family (convergence), and (ii) within what plethodontid tribe Bolitoglossini. Because of the surprising is currently recognized as a single taxon (parallelism). These evo- results, several populations of the in-group and Pseudoeurycea lutionary events are of adaptive significance because to invade the were collected on repeated field trips and sequenced indepen- lowland tropics salamanders must be either arboreal or fossorial; dently. DNA was extracted following standard techniques (4). the repeated evolution of elongation and attenuation has led to We obtained 544 bp of the large 16S subunit ribosomal mtDNA multiple lowland invasions. (5), 569 bp of the cytochrome b gene (6), and 709 bp of the ND4 gene (7). Amplification was done via the PCR (8) using the ropical plethodontid salamanders form a monophyletic clade primers 16Sar and 16Sbr (9) for 16S, MVZ15 and MVZ18 (10) Tof more than 200 species that display diverse body forms, for cytochrome b, and ND4 (7) and MVZ112 (3) for the ND4 including extreme elongation accompanied by limb reduction gene. Following standard techniques (4), cycle-sequencing prod- associated with fossorial habits (1). All lowland tropical pleth- ucts were purified by using ethanol precipitation and separated odontids are either fossorial or arboreal, and fossoriality has by electrophoresis on a 6% polyacrylamide gel using an ABI 377 been an important factor in the invasion of lowlands by these DNA sequencer (Applied Biosystems). Sequences were read more typically cool-adapted upland . The fossorial Oe- from both strands and aligned to each other by eye in the dipina has many species that range south and east of the Isthmus program SEQUENCE NAVIGATOR version 1.0.1 (Applied Biosys- of Tehuantepec, from southern Me´xico into northwestern South tems). Sequence divergences were estimated by using the America; these differ from all other tropical plethodontids in Kimura two-parameter distance (11). Phylogenetic analyses having 18–22 rather than an invariable 14 trunk vertebrae. The were performed by using PAUP 4.0b2a (D. Swofford, Smithsonian other fossorial lineage, the equally attenuate and elongate Institution, Washington, DC). Parsimony analysis used heuristic Lineatriton lineolus, is found only in restricted parts of eastern searches by stepwise random addition of taxa, with 20 replica- Me´xico, north of the Isthmus of Tehuantepec. Oedipina evolved tions and tree bisection and reconnection branch swapping with elongation from a more generalized morphology by increasing the MULPARS option in effect. Zero-length branches were col- the number of body segments, and hence vertebrae, a develop- lapsed. Both the consistency index (12), and retention index (13) mental mechanism that has evolved repeatedly within were calculated. We used two character weighting schemes: salamanders as a whole, and even within the clade (Bolito- equal weighting for all codon positions, and differential weight- glossini) to which the tropical species belong. Lineatriton,in ing in which we downweighted third codon position transitions contrast, is unique among all salamanders in having evolved an entirely different developmental mechanism. Its trunk vertebrae Data deposition: The sequences in this paper have been deposited in the GenBank database number only 14, the same as in all other tropical salamanders (accessions nos. AF379643–AF379681 and AF380754–AF380831). except for Oedipina, but the individual vertebrae are narrow and *Present address: Museum of Comparative Zoology, Harvard University, Cambridge, MA elongate. As a consequence, the external form of L. lineolus and 02138. its adaptation for fossorial existence are so similar to those of †To whom reprint requests should be addressed at: Museum of Vertebrate Zoology, 3101 species of Oedipina that it was included in the genus Oedipina Valley Life Sciences Building, University of California, Berkeley, CA 94720-3160. E-mail: until 1950 (2). [email protected].

7888–7891 ͉ PNAS ͉ July 3, 2001 ͉ vol. 98 ͉ no. 14 www.pnas.org͞cgi͞doi͞10.1073͞pnas.131203598 Downloaded by guest on September 27, 2021 to a ratio of 1:0.16 transversion͞transition. The transversion to transition ratio was estimated by maximum likelihood analysis. Neighbor-joining reconstructions (14) used Kimura two- parameter distances. Maximum likelihood analysis used the heuristic algorithm, empirical base frequencies, and the Hase- gawa-Kishino-Yano (15) model of character evolution. Decay indices and bootstrap values in excess of 50% are reported (1,000 pseudoreplicates for parsimony analysis with heuristic search and simple addition of taxa). Morphological comparisons are based on study of proportions of preserved individuals, cleared and stained skeletal prepara- tions, and radiographs of preserved specimens. Results The three genes studied each display extensive differentiation among populations of L. lineolus, with Kimura two-parameter distances as great as 18% (cytochrome b), 16% (ND4), and 5% (16S). These levels are much greater than typically found within other species of bolitoglossine salamanders (e.g., ref. 4), or other taxa (16). Independent and combined data analyses are consis- tent in finding mitochondrial para- or polyphyly of Lineatriton. Mitochondrial haplotypes do not form a monophyletic group but rather fall out in different parts of the mtDNA gene tree. A maximum parsimony analysis of a combined data set using equal weighting produced 80 equally parsimonious trees (L ϭ 2,183 steps; 545 characters were parsimony informative; consistency index ϭ 0.478, retention index ϭ 0.763). In this analysis (Fig. 1), populations of L. lineolus are deeply nested within Pseudoeury- cea, and samples of Lineatriton are not monophyletic. Samples of L. lineolus from southeast Veracruz (population 1, Los Tuxtlas) form a clade that is sister to the closely related Oaxacan species Pseudoeurycea werleri and Pseudoeurycea mystax. A well sup- ported clade consisting mainly of samples of is the sister taxon of a clade that includes samples of L. lineolus from central western Veracruz (populations 2 and 3, Cerro Chicahuaxtla and Barranca de San Miguel). We recover two well supported clades (clade 1, bootstrap value 96%, decay index 10, and clade 2, bootstrap value 77%, decay index 4; Fig. 1), each containing Lineatriton, and these clades are deeply Fig. 1. (Left) Consensus tree of maximum parsimony analysis, combined data nested within Pseudoeurycea. When all samples of L. lineolus are set with equal weighting. (Right) Maximum likelihood phylogram of com- forced to be monophyletic, the most parsimonious tree is 17 steps bined data set. For the parsimony tree bootstrap values (above lines) and longer than the most parsimonious tree overall. A Wilcoxon decay indices (below lines) for relevant nodes. Shown are four individuals each signed-ranks test (17) indicates that these two trees differ for three populations currently assigned to L. lineolus: L. lineolus 1 (red) from significantly (n ϭ 58, P ϭ 0.0356), thus monophyly of L. lineolus the slopes of Volca´n San Martı´n,Los Tuxtlas, Veracruz, Me´xico; L. lineolus 2 is rejected. Independent and combined data sets with different (yellow) from Barranca de San Miguel, Fortı´nde las Flores, Veracruz, Me´xico; and L. lineolus 3 (yellow) from slopes of Cerro Chicahuaxtla, Cuautla´pan, weighting schemes resulted in topologies identical to that ob- Veracruz, Me´xico. Other samples used in analysis include (all from Me´xico tained in the equally weighted analysis, with minor differences in except an outgroup, Batrachoseps, from California): P. werleri, Volca´n San the bootstrap support for clades. The topology obtained in the Martı´n, Los Tuxtlas, Veracruz; P. mystax, near Ayutla, Oaxaca; P. leprosa 1, maximum likelihood analysis is nearly identical to the parsimony Xometa, Veracruz; P. leprosa 2, east of Santa Marta, Me´xico; P. leprosa 3, Las analysis topology (Fig. 1, ϪLn ϭ 11,928.72), the only difference Vigas, Veracruz; P. leprosa 4, Tres Mogotes, Puebla; P. leprosa 5, La Malinche, being that this analysis provides more structure for relationships Puebla; P. leprosa 6, Tres Mogotes; P. sp. nov. 1, near Chiconquiaco, Veracruz; EVOLUTION at the base of the tree, within Pseudoeurycea. Pseudoeurycea juarezi, Cerro Pelo´n, Oaxaca; Pseudoeurycea saltator,LaEs- peranza, Oaxaca; Pseudoeurycea unguidentis, Cerro Machı´n,Oaxaca; Pseudo- Discussion eurycea longicauda, Lengua de Vaca, Me´xico; Pseudoeurycea altamontana, east of Santa Marta, Me´xico; Pseudoeurycea robertsi, Raı´ces, Me´xico; P. sp. The phylogenetic hypotheses derived from the mitochondrial nov. 2, Tlaxiaco, Me´xico; Pseudoeurycea tenchalli, Filo de Caballo, Guerrero; data unambiguously reject the monophyly not only of Lineatriton Pseudoeurycea cochranae, San Pedro el Alto, Oaxaca; P. smithi, Cerro San but also of L. lineolus. This is extraordinary because the mor- Felipe, Oaxaca; P. sp. nov. 3, Cerro Pelo´n, Oaxaca; Thorius troglodytes, Puerto phology (Figs. 2 and 3) of the single recognized species is del Aire, Veracruz. extreme in its degree of specialization (miniaturization, elonga- tion, and attenuation of body, tail and limbs), and has been considered to be unique in the combination of characters thus our comparison was inappropriate (18), (ii) ancestral mi- (especially with respect to elongation of the vertebrae) within the tochondrial polymorphisms exist (19), and (iii) local hybridiza- order Caudata (1). The evolutionary implication of our phylo- tion and subsequent introgressive gene flow has occurred (20). genetic hypothesis is that the extreme morphology of the dif- We consider these in order. ferent populations of L. lineolus has been acquired indepen- The results from independent analyses of each of the three dently from ancestors with generalized morphology. genes are consistent with the analyses of all data combined in Alternative explanations for our findings include: (i) copies of revealing the mitochondrial paraphyly of Lineatriton. Such con- the sequenced genes have been transposed to the nucleus and gruence between the three genes would be unlikely if paralogous

Parra-Olea and Wake PNAS ͉ July 3, 2001 ͉ vol. 98 ͉ no. 14 ͉ 7889 Downloaded by guest on September 27, 2021 Fig. 3. Photographs of species studied together with simplified tree, show- ing body form and two levels of homoplasy with respect to elongation, Fig. 2. Ideograms showing the relationship between attenuation and ver- attenuation, and fossorial specialization in tropical salamanders. The first is tebral proportions for individuals of three species of plethodontid convergence for elongation related to fossorial life shown in comparisons of salamanders. Three specimens displaying different morphologies are shown. Lineatriton and Oedipina, while the second is parallelism with respect both to Each was radiographed to show the skeleton. (Left) Oedipina stenopodia from mode of development for elongation and adaptation for fossorial living Guatemala. (Center) L. lineolus from Me´xico. (Right) P. leprosa from Mexico. shown for the paraphyletic taxon Lineatriton.(A) L. lineolus from Los Tuxtlas, The two elongate species have the same head ϩ body length, and the major Veracruz, Me´xico. (B) P. mystax from Ayutla, Oaxaca, Mexico. (C) L. lineolus difference between them is that the vertebrae of Lineatriton are longer than from Cerro Chicahuaxtla, Veracruz, Me´xico. (D) P. leprosa from slopes of Pico those of Oedipina, which are about the same length as those of Pseudoeury- de Orizaba, Veracruz, Me´xico. (E) P. robertsi from near Raices, Me´xico, Me´xico. cea, a shorter-bodied form with a head slightly larger than the other two (F) Oedipina uniformis from slopes of Volca´n Irazu´, San Jose´, Costa Rica. individuals illustrated. Every fifth vertebra is shaded, to better display the effect of vertebral lengthening. Each species has a single cervical vertebra (the atlas) and a single sacral vertebra; there are 20 trunk vertebrae in Oedipina but so it is unlikely that the paraphyly of the two populations of only 14 in Lineatriton and Pseudoeurycea. The transverse processes of the Lineatriton can be explained by the presence of nuclear copies of sacral vertebra of Lineatriton are located in an extreme posterior position, and the mtDNA genes. they are directed posteriorly, effectively adding to the relative interlimb Retention of polymorphisms (19) is highly unlikely given the distance as compared with the other species. The morphology displayed by long period since the splitting of the relevant lineages. Using a Oedipina is a common homoplasy in plethodondontid salamanders, seen for cytochrome b calibration (20, 21) for North American salaman- example in the distantly related bolitoglossine genus Batrachoseps from California. drids, the estimated divergence time between the populations of Lineatriton from central Veracruz and P. leprosa group (clade 1) is about 20–25 million years, and between Lineatriton from Oaxaca and P. werleri (clade 2) is about 16–18 million years. copies of either gene were sequenced (18). Nuclear copies are Hybridization with sympatric forms is unlikely, given that under different mutational constraints and are expected to show Lineatriton is a highly specialized burrower. The mechanics of different evolutionary patterns compared with the authentic courtship (22) argue against mating between species with ex- mitochondrial genes. Nucleotide substitutions occur more ran- tremely different morphology. Furthermore, hybridization domly in nuclear genes, and nuclear insertions show less marked has been demonstrated only once within the entire tropical bo- transition and codon position biases than is the case for mito- litoglossine clade, and that occurred between two close rela- chondrial genes (18). Analysis of molecular evolution of the tives with different ecologies after habitat modification by mitochondrial genes shows that the genes sequenced have a humans (23). typical ‘‘mitochondrial’’ behavior, e.g., most variable sites are in High bootstrap support and high decay indices in critical parts the third codon position as is typical for protein coding regions, of the tree show that our phylogenetic hypothesis is well resolved.

7890 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.131203598 Parra-Olea and Wake Downloaded by guest on September 27, 2021 Accordingly, the phylogeny recovered from our analysis of we suspect that several additional species eventually will be mtDNA probably reflects the organismal phylogeny. recognized in this limited area alone. Nonsister lineages, at present considered to be populations of The adaptive significance of the ‘‘Lineatriton’’ phenomenon L. lineolus, share the derived morphological features previously appears to be local and limited. Given the fact that only one thought to have been uniquely derived, but which we believe clade, Oedipina, of the large number of tropical bolitoglossine evolved twice independently (Fig. 2). We infer, on grounds of clades has evolved an escape from the constraint of a fixed parsimony, that the ancestral morphology of the clade that number of trunk vertebrae, it might seem reasonable to expect contains the two subsets of L. lineolus, as well as P. leprosa, P. the adaptive radiation of tropical salamanders to have exploited werleri and P. mystax, is that of nonfossorial Pseudoeurycea (the the mode of evolution used by Lineatriton. However, only in three terrestrial species mentioned appear typical of the modal Veracruz has this option been used, although it appears to have morphology in the genus). The possibility that the ancestral evolved twice. In contrast, the developmental option used by morphology of this clade is that found in Lineatriton would Oedipina, apparently difficult to achieve in the large tropical require that the morphology found in P. leprosa, P. werleri, and bolitoglossine clade because it has only evolved once (Oedipina P. mystax be secondarily derived, i.e., an evolutionary reversal to is a well supported monophyletic clade, ref. 4), has been more the condition typical of distantly related species of Pseudoeury- successful, as evidenced by the wide geographic range (from cea, which would require multiple morphological reversions. Chiapas throughout Central America to Colombia and Ecua- Examples of unusual morphological specialization include the dor), great elevational range (from sea level to over 2,500 m), ribs of Lineatriton, which are greatly reduced in size (24), the rib and large number of species (more than 20). Although appar- heads and rib bearers, which are specialized in structure (2, 24), ently adaptive advantages followed the evolution of increased and the transverse processes of the sacral vertebrae, which are numbers of trunk vertebrae in ancestral Oedipina, no adaptive located in an unusual position near the end of the centrum (Fig. radiation or burst of species formation followed vertebral elon- 2) and are oriented more posteriorly than laterally. Although it gation, and the salamanders displaying it can only be considered is difficult to imagine a restoration of the ancestral conditions terminal twigs in the bolitoglossine radiation, which encom- from these extremes, it is also difficult to understand how these passes about half of the living species of salamanders. several features could have evolved in parallel. The populations we studied that currently are referred to L. We thank M. Garcı´a-Parı´s, J. Hanken, F. Pedroche, M. Lo´pez, M. Cero´n, lineolus belong to at least two species that are very similar C. Cero´n, and L. F. Garcı´a for assistance in the field, herpetological morphologically. The type locality in the original description is collections at Universidad Autonoma de Me´xico (L. Canseco-Marquez, vague (Mexican Tableland, ref. 25), but is more appropriate to O. Flores-Villela) for samples, Secretarı´a de Medio Ambiente y Recur- the populations from central western Veracruz than from the sos Naturales for providing collecting permits, and M. Garcı´a-Parı´s, J. Hanken, J. Patton, M. Slatkin, J. Rodrı´quez-Robles, S. Kuchta, and M. Los Tuxtlas area. Complicating species determination is the fact Wake for discussion. A. Summers provided valuable advice and created that in central western Veracruz (populations 2 and 3) diver- Figs. 2 and 3. Studies were financed in part by grants from the U.S. gences in the three genes sequenced are so great (greatest National Science Foundation and the National Geographic Society. Kimura two-parameter distances for the three genes in this area G.P.-O. was sponsored by a fellowship from Consejo Nacional de Ciencia are 13% for cytochrome b, 9% for ND4, and 1.6% for 16S) that y Tecnologı´a.

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Parra-Olea and Wake PNAS ͉ July 3, 2001 ͉ vol. 98 ͉ no. 14 ͉ 7891 Downloaded by guest on September 27, 2021